PTHrP (parathyroid hormone-related protein) was first identified as the factor responsible for the paraneoplastic syndrome of humoral hypercalcemia of malignancy. The most abundant physiological source of PTHrP is milk, and data indicate that PTHrP produced in the lactating mammary gland (MG) is released into the circulation. Our central hypothesis is that PTHrP released by the MG acts systemically to liberate skeletal Ca for milk production, and that PTHrP acts locally to promote Ca transport across mammary epithelial cells (MEC). The effect of PTHrP gene knockout on lactation has heretofore been unexplored because PTHrP is required for MG development. However, to circumvent this problem and in order to test our hypothesis, we will use a cre/lox system to generate mice with a conditional deletion of the PTHrP gene specifically in MEC during pregnancy and lactation. We will then look for decreases in milk Ca, and in the putative lactation-associated systemic effects of PTHrP in these mice. These effects include decreased bone density, increased bone turnover, and increased phosphate and cAMP in urine. We will use a novel in vitro system based on "mammospheres" to investigate the role of PTHrP in Ca transport in MEC. Using 45Ca as a tracer, we will determine which regions of PTHrP, if any, are important for Ca transport across MEC. Finally, PTHrP and the calcium-sensing receptor (CaR) have been shown to regulate placental calcium transport. We will determine if a similar interplay of PTHrP and the CaR involved in regulating Ca metabolism and transport during lactation. Whether PTHrP mediates mobilization and transport of Ca in lactation is a long-standing unanswered question. We believe the proposed research is the most direct and informative approach to finally understand the underlying reasons for the abundance of PTHrP in milk.